Infectious DNA clones

12 February 2009

img_0420The development of recombinant DNA methods by Cohen and Boyer in 1973, together with the discovery of reverse transcriptase by Temin and Baltimore in 1970, made it possible to introduce a mutation at any location in a viral genome. The essential reagent is an infectious DNA clone, a double-stranded DNA copy of the viral genome carried in a bacterial plasmid. These DNAs (or RNAs produced from them) can be introduced into cells by transfection1 to produce infectious virus.

Infectious clones of viral genomes were initially produced in the late 1970s and early 1980s. The first was made in 1978 by inserting a DNA copy of the RNA genome of the bacteriophage QB, made with reverse transcriptase, into a plasmid vector. Infectious virus was produced when the cloned viral DNA was inserted into E. coli. In 1980,  infectious cloned retroviral DNA was produced by inserting the integrated viral DNA from the cellular genome into a plasmid vector. The next year, a DNA copy of the RNA genome of poliovirus was produced by reverse transcription and inserted into a plasmid vector. When the cloned copy of the viral genome was introduced into mammalian cells, infectious virus was produced.

Since these early findings, infectious DNAs of members of nearly every virus family have been reported. Some have been more difficult to produce than others. For example, to recover infectious influenza virus from cloned DNA, an expression system is used in which cloned DNA copies of the eight RNA segments are flanked by two promoters. Upon introduction of the eight plasmids into cultured cells, two types of RNAs are produced: mRNAs for the synthesis of viral proteins, and viral RNAs for replication and incorporation into virions. The production of infectious DNAs of  (-) strand RNA viruses was counterintuitive. The (-) strand genomic RNA of these viruses is not infectious because it cannot be translated or copied into mRNA in the cell. In the first attempts, full-length (-) strands, produced by in vitro transcription of cloned DNA, were introduced into cells that produce the proteins required for mRNA synthesis. However, no infectious virus was recovered. The solution, found first with rabies virus, was to transfect full-length (+) strand RNA into cells that produce the viral nucleocapsid protein, phosphoprotein, and polymerase. In these cells, the (+) strand RNA is copied into (-) strand RNAs which then initiate an infectious cycle.

The double-stranded RNA genome of reoviruses is not infectious because it cannot be translated. To produce an infectious clone, DNA copies of the genome segments are placed in plasmids under the control of a T7 RNA polymerase promoter. When all 10 plasmids are introduced into cells that synthesize T7 RNA polymerase, viral mRNAs are produced which initiate an infectious cycle.

The complete genomes of many DNA viruses, including polyomaviruses, papillomaviruses, and adenoviruses, are sufficiently small to be carried in plasmid vectors. However, conventional plasmid vectors cannot accommodate the larger DNA genomes of herpesviruses and poxviruses; therefore cosmids and bacterial artificial chromosomes vectors, which can accept larger inserts, have been used. Such vectors have also been used to carry DNA copies of the largest RNA genomes, those of members of the Nidovirales. Poxvirus DNA is not infectious, because cellular DNA-dependent RNA polymerase cannot recognize the viral promoters. Viral DNA-dependent RNA polymerase and transcription proteins must therefore be provided.

The infectious viral DNA clone is a double-edged sword.  It enables manipulation of the viral genome at will, allowing unprecedented genetic analysis and the use of viruses as vectors for gene therapy. But nearly any virus can now be recovered from the nucleotide sequence – effectively making it impossible to ever truly eradicate a virus from the globe.

1The introduction of DNA or RNA into cells with the object of obtaining infectious virus is called transfection (transformation-infection). This phrase was originally coined to describe production of bacteriophage lambda after transformation of cells with viral DNA. Transfection is now incorrectly used to describe the introduction of any DNA into cells. This usage has come about to avoid confusing DNA-mediated transformation with the process of oncogenic transformation.

Taniguchi T, Palmieri M, & Weissmann C (1978). A Qbeta DNA-containing hybrid plasmid giving rise to Qbeta phage formation in the bacterial host [proceedings] Annales de microbiologie, 129 B (4), 535-6 PMID: 754572

Lowy DR, Rands E, Chattopadhyay SK, Garon CF, & Hager GL (1980). Molecular cloning of infectious integrated murine leukemia virus DNA from infected mouse cells. Proceedings of the National Academy of Sciences of the United States of America, 77 (1), 614-8 PMID: 6244569

Racaniello, V., & Baltimore, D. (1981). Cloned poliovirus complementary DNA is infectious in mammalian cells Science, 214 (4523), 916-919 DOI: 10.1126/science.6272391

Schnell MJ, Mebatsion T, & Conzelmann KK (1994). Infectious rabies viruses from cloned cDNA. The EMBO journal, 13 (18), 4195-203 PMID: 7925265

KOBAYASHI, T., ANTAR, A., BOEHME, K., DANTHI, P., EBY, E., GUGLIELMI, K., HOLM, G., JOHNSON, E., MAGINNIS, M., & NAIK, S. (2007). A Plasmid-Based Reverse Genetics System for Animal Double-Stranded RNA Viruses Cell Host & Microbe, 1 (2), 147-157 DOI: 10.1016/j.chom.2007.03.003

  • Michael Day

    I am just beginning to anticipate needing to produce infectious clones of reovirus and rotavirus, and I had not even begun to look at the available literature. The Kobayashi reference is going to help immensely, I predict!
    (Just found your blog and I have enjoyed reading it…)

  • http://www.virology.ws profvrr

    What I did not mention in the post was that many labs tried for years
    to produce infectious DNA of reovirus. All kinds of reasons for why it
    didn't work were set forth. In the end, it turned out that the
    sequences were wrong. Kobayashi et al sequenced many clones until they
    got it right, and the result was infectivity. So make sure your
    sequences are correct….sequence multiple independent clones for each
    dsRNA segment and arrive at a consensus. Good luck, and glad you enjoy
    virology blog.

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  • Jun

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  • Donutsgalore

    A little late to the party, but here: http://www.ncbi.nlm.nih.gov/pubmed/21798351

  • Doraemon el gato cósmico

    So basically this is how they created HTLV-3 (HIV), cloning MVV & BIV with the possibility of EIAV? Everything makes sense. It was so easy.

  • Doraemon el gato cósmico

    I too am a Little late here but I am fasinated about the origins of HTLV-3, WOW, does this all start to make perfect sense! So basically HTLV-3 could be the ancestor of many SIV´s not the other way around! This is scary stuff!